Evaluation of antioxidant properties of monoaromatic derivatives of pulvinic acids

Article abstract of DOI:10.1021/jm801500h

The natural mushroom pigment Norbadione A and three other pulvinic acids were shown by our group to display very efficient antioxidant properties by comparison with a collection of potent molecules including catechols, flavonoids, stilbenes, or coumarins.

Full text of DOI:10.1021/jm801500h

2454
J. Med. Chem. 2009, 52, 2454–2464
Evaluation of Antioxidant Properties of Monoaromatic Derivatives of Pulvinic Acids
Damien Habrant,‡ Ste´phane Poigny,§ Muriel Se´gur-Derai,§ Yves Brunel,§ Benoˆıt Heurtaux,| Thierry Le Gall,| Axelle Strehle,
Re´gis Saladin, Ste´phane Meunier,‡,* Charles Mioskowski,†,‡ and Alain Wagner‡
Laboratoire de Synthe`se Bio-Organique, CNRS-UMR 7175/LC1, Institut Gilbert Laustriat, Faculte´ de Pharmacie, Illkirch, 67401 France,
Centre de Recherche et de De´Veloppement Pierre Fabre Dermo-Cosme´tique, 17 Alle´e Camille Soula, 31320 Vigoulet-Auzil, France, CEA,
iBiTecS, SerVice de Chimie Bioorganique et de Marquage, Baˆt. 574, 91191 Gif-sur-YVette, France, PhytoDia, Poˆle API, BouleVard Se´bastien
Brant, 67412 Illkirch, France
ReceiVed NoVember 28, 2008
The natural mushroom pigment Norbadione A and three other pulvinic acids were shown by our group to
display very efficient antioxidant properties by comparison with a collection of potent molecules including
catechols, flavonoids, stilbenes, or coumarins. Despite numerous publications on robust and straightforward
synthetic access to pulvinic acids by us and others, no report has been made to unravel the structure-activity
relationships that govern the striking antioxidant activity. Herein is presented the synthesis of 18 diverse
pulvinic acid derivatives and the study of their radical scavenging capacities by four different assays. The
influence of each of the two phenyl rings, of their substituents and of the lateral chain on the antioxidant
properties, was explored to reveal a simplified structure of excellent activity. These results, along with the
absence of cytotoxicity, make the synthesized compounds interesting to evaluate for several biological
activities and especially for anti-inflammatory effects and skin protection against UV induced oxidative
stress.
Introduction
Reactive oxygen species (ROS)a are natural products of
metabolic processes in an aerobic environment and can be
generated by different pathways.1,2 Examples of ROS species
are hydrogen peroxide, hydroxyl radicals, or superoxide anion.
They are produced by sources such as bioreductively activated
molecules, UV exposure, or by low-molecular-weight complexes
of transition state metals such as iron via the Fenton reaction.
Organisms can normally defend themselves against these highly
reactive species using enzymes such as superoxide dismutase,
catalase, and glutathione peroxidase, or by nonenzymatic
mechanisms.2,3 The latter involve organic molecules called
antioxidants, such as vitamin C, vitamin E, or glutathione,
capable of scavenging the oxidant species by hydrogen donation
or breaking of an oxidation propagation chain.4 Imbalances in
the detoxification of ROS relative to their production, due to
either an abnormal production of ROS or depletion of antioxi-
dant defenses lead to the commonly called oxidatiVe stress state.
For many years, this state has been known to be implicated in
the etiology of a large number of diseases and disorders2,5 such
as cancer,6 coronary heart disease,7 or rheumatoid arthritis.8
Enhanced free radicals production and oxidative damage are
also implicated in aging1a and related neurodegenerative diseases
such as Alzheimer’s,9 Parkinson’s,10 or Huntington’s diseases.11
Thus finding therapeutic agents that can scavenge ROS is a high
priority in medical research today, and many therapeutic
strategies using antioxidant have already been reported in the
Figure 1. Structure of Norbadione A (left) and di-O-methylatromentic
acid (right).
literature or patented with promising in vivo results.12 In this
context, our group described a high-throughput screening
method to search for antioxidant molecules in natural extracts.
This strategy is based on competitive immunoassay techniques
to rapidly evaluate the ability of different extracts or compounds
to protect thymidine under different oxidative stresses.13,14
Norbadione A (NBA) (Figure 1, left), a mushroom pigment
isolated from the two mushrooms Xerocomus badius and
Pisolithus tinctorius,15 was shown to be a potent protector of
thymidine under both γ irradiation and UV exposure in the
presence of H2O2. Unfortunately, the pro-oxidant effect of NBA
was observed on a plasmid-DNA under the oxidative conditions
of a Fenton-like system (FeSO4/H2O2/EDTA). Three structurally
related pulvinic acids and especially di-O-methylatromentic acid
(Figure 1, right) were then evaluated and shown to display
antioxidant properties higher than that of a collection of
flavonoids and similar to NBA. Furthermore, the ROS-scaveng-
ing properties of di-O-methylatromentic acid were confirmed
in DNA protection experiments, showing no pro-oxidant effect.
This pulvinic acid possesses a single hydroxyl function, which
makes its structure peculiar by comparison with other potent
antioxidants that were assayed. Indeed, the high efficiency of
* To whom correspondence should be addressed. Phone: 33 3 90 24 42
95. Fax: 33 3 90 24 43 06. E-mail: meunier@bioorga.u-strasbg.fr. Address:
Faculte´ de Pharmacie, 74 Route du Rhin BP24, F-67401 Illkirch, France.
†
Deceased June 2nd, 2007. This communication is dedicated to the
memory of Charles Mioskowski who initiated this work.
‡
Laboratoire de Synthe`se Bio-Organique, Institut Gilbert Laustriat.
Pierre Fabre Dermo-Cosme´tique.
Service de Chimie Bioorganique et de Marquage, CEA.
§
|
PhytoDia.
a Abbreviations: ROS, reactive oxygen species; NBA, norbadione A;
dThd, thymidine.
10.1021/jm801500h CCC: $40.75
2009 American Chemical Society
Published on Web 03/23/2009

Monoaromatic DeriVatiVes of PulVinic Acids
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2455
pulvinate 8b, deprotection of the methyl enol ether was achieved
in 65% yield without significant deprotection of the aromatic
methoxy group, as described on pinastric acid.24
Type II products 2a and 2c were prepared as shown in
Scheme 2. Tetronic derivative 9 was prepared using described
procedures25 and protected as benzyl ether under Mitsunobu
conditions (74%). Deprotonation of 10 using LDA and subse-
quent addition of ketoesters 11 provided alcohols 12 (66-69%),
which were dehydrated using the conditions described above,
albeit in lower yields (18-44%, nonoptimized). For 13a, only
the E-isomer was obtained, while for 13c, both E and Z isomers
were obtained as a mixture (ratio 75/25 by NMR), the E-isomer
being isolated by column chromatography. E-13 were then
subjected to hydrogenolysis, affording type II products 2a and
2c (48 and 66% yield, respectively).
Figure 2. General structure of pulvinic acids and monoaromatic
targeted molecules.
Then, the monoaromatic pulvinic dilactone 14, whose syn-
thesis has previously been described by our group,26 was used
in order to modify the nature of the exo-cyclic double-bond
functionalization (Table 1). By heating under reflux an alcoholic
solution of 14, the nonregioselective opening of the dilactone
moiety led to a separable mixture of type I and type II ester
adducts. Using methanol, type I product 1b was obtained in
54% yield, type II adduct 2b in 44% yield (Table 1, entry 1);
similar results were obtained using isopropyl alcohol, affording
products 1g and 2d (Table 1, entry 2). Using other nucleophiles,
type I adducts could be obtained selectively. With dibutylamine
and morpholine, the TBAF-mediated ring opening of 1426 gave
access to type I amides 1h and 1i, respectively, in excellent
yields (Table 1, entries 3 and 4). In the absence of TBAF, the
opening of 14 by 1,3-diaminopropane and N,O-dimethylhy-
droxylamine led solely to type I adducts 1j and 1k in good yields
(Table 1, entries 5 and 6, respectively). Such regiospecificity
in the absence of TBAF by comparison with the reactivity of
simple amines is not explained yet. Finally, using methyllithium
(Scheme 3), only type I alcohol 1l could be isolated with a good
yield. We assume this product arose from a two-step sequence:
(1) nucleophilic opening of 14 by a first equivalent of methyl
anion, leading to the formation of a ketone, (2) attack of a second
equivalent of methyl anion on the ketone.
flavonoids,16 stilbenes,17 or coumarine derivatives18 is known
to arise from their polyhydroxylated structures.
Therefore, pulvinic acid derivatives could represent original
and promising candidates as new antioxidant agents. This
prompted us to further investigate this class of compounds. To
efficiently explore structure-activity relationships (SARs), we
based the design of the pulvinic derivatives on the known
essential parameters that govern antioxidant activity of phenolic
compounds. Especially it is well established that an extended
aromatic structure is favorable in order to stabilize the oxygen
radicals formed. For this reason, simplified analogues bearing
only one aromatic ring such as type I and II compounds were
designed (Figure 2). Aromatic ring substitution on phenols,
particularly in the ortho and the para position, also plays a
crucial role in the O-H bond dissociation enthalpy (BDE) as
well as in the ionization potential (PI).19 In the present study,
the para substitution exploration was preferred for synthetic ease
(Figure 2). Finally, variations of the lateral function R1 in
pulvinic derivatives should allow us to explore a possible effect
of the intramolecular hydrogen bond between the enolic function
and the carbonyl group.
We describe herein 12 examples of type I compounds, four
examples of type II molecules, and two examples of symmetric
pulvinic acids as well as their ROS-scavenging ability with
regard to the study of the protection of thymidine and DNA
and the scavenging of the superoxyde anion, along with
cytotoxicity results.
Symmetrical pulvinic acid derivatives 3a,b were obtained by
following procedures described by Le Gall and Mioskowski
(Figure 3).27 Interestingly, both type I adducts 1 and symmetrical
pulvinic derivatives 3 are striking yellow or orange solids, while
type II products 2 are pale-yellow or white solids.
Chemistry
Biological Results
The synthetic route followed to obtain type I compounds 1a-f
is summarized in Scheme 1. Addition of zinc enolate of methyl
propionate on methoxymaleic anhydride 420 occurred regiose-
lectively on the carbonyl adjacent to the methoxy group, as
described by Pattenden on 2-aryl-3-methoxymaleic anhydrides,21
to give alcohol 5 (51%) as a mixture of two diastereoisomers
(70/30 ratio determined by 1H NMR). Dehydratation of 5 using
trifluoroacetic anhydride under basic conditions yielded alkene
6 (90%) in a 90/10 mixture of E- and Z-isomers.22 The E-isomer
could be isolated by column chromatography, and the following
transformations were performed on this isomer. Compound 6
was iodinated using iodine and cerium(IV) ammonium nitrate21
to give 7 (94%). Then a poly ionic gel supported Pd catalyst,23
reported in our group, was used to perform Suzuki-Miyaura
cross-coupling reactions with various boronic acids. This
heterogeneous catalyst allowed the efficient transformation of
7 into pulvinates 8a-f (58-78%). Finally, deprotection of
compounds 8 using 1 equiv of boron tribromide yielded the
type I products 1a-f (58-85%). Interestingly, in the case of
The antioxidant activity of the synthesized pulvinic derivatives
was determined using four different assays. Activity was first
measured using immunoenzymatic assays, based on the study
of the degradation of thymidine, under either UV-exposure or
Fenton stress. The compounds were also tested for the protection
of plasmidic DNA under Fenton stress. Finally, the radical
scavenging abilities of superoxide anion radical were studied
as well as the cytotoxicity of these new products. Structures of
the tested molecules are summarized in Table 2.
Protection of Thymidine. This test is based on the degrada-
tion of thymidine (dThd) by oxidative stress under aerobic
conditions.13,14 The unmodified thymidine remaining after this
degradation step is quantified by a competitive enzyme immu-
noassay by using a specific antithymidine antibody. Two
oxidative conditions were tested, using either a UV irradiation
at 254 nm (1.75 J/cm2) of a hydrogen peroxide solution buffered
at physiological pH (Figure 4A) or a Fenton-like system, that
is, in the presence of Fe2+/EDTA and hydrogen peroxide (Figure

2456 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Habrant et al.
Scheme 1a
a Reagents and conditions: (a) Methyl propionate, LDA, ZnCl2, THF, -78 °C, 45 min then 4, 1 h; (b) TFAA, Et3N, DMAP, DCM, 0 °C to rt, 12 h; (c)
I2, CAN, MeCN, 40 °C, 3 h; (d) ArB(OH)2, supported Pd catalyst, iPr2NH, MeCN, H2O, 85 °C, 6 h 30 min; (e) BBr3, DCM, 0 °C, 2 h.
Scheme 2a
a Reagents and conditions: (a) BnOH, DIAD, PPh3, THF, rt, 12 h; (b) LDA, THF, -78 °C, 45 min then 11, 1 h; (c) TFAA, Et3N, DMAP, DCM, 0 °C,
12 h; (d) H2, Pd/C, DCM, rt, 12 h.
4B). Type I and type II products were evaluated, as well as
pulvinic acids, NBA and Trolox, as a reference antioxidant. Each
compound was assayed twice at a single concentration, either
100 µM for the UV/H2O2 test or 500 µM for the Fenton test.
The screening results presented in Figure 4 were confirmed by
the determination of EC50 values for six selected compounds:
1b, 1h, 1l, 2a, 2b, and 3b. The EC50 values were found to be
in very good agreement with the data presented in Figure 4
(see Supporting Information for EC50 values and dThd protection
data).
The differences in protections observed between the dThd
assays under UV/H2O2 and Fenton-like conditions reflect the
differences in the oxidative species generated in the system.
Indeed, in the UV/H2O2 test, HO• radicals, generated by
homolytical cleavage of the oxygen-oxygen bond of H2O2
under UV irradiation, are probably the main species responsible
for the thymidine degradation. Therefore and as previously
discussed,13 the protective effects measured by this assay are
closely related to the HO• radical-scavenging abilities of the
tested compounds. On the other hand, in the case of the Fenton
test, the formation of high-valence iron-oxo species is expected,
along with other radical species.28 Some molecules can strengthen
the Fenton-like oxidative system and therefore display a pro-
oxidant behavior, which can not be evaluated in the UV test.14,15
These differences are clearly highlighted by our references, NBA
and Trolox, which exhibit opposite activities in these two
conditions (NBA being highly active in the UV/H2O2 test and
not in the Fenton test, Trolox active in the Fenton test and not
in the UV/H2O2 test). Indeed NBA is reported to be a potent
HO• scavenger but also to display pro-oxidant properties in the
presence of iron.13,14
The observation of good protections in both tests for many
of the assayed compounds, especially type I derivatives 1 and
symmetrical pulvinic acids 3, demonstrate that these molecules
are efficient protectors in different oxidative stress conditions
and indicate that they probably do not display pro-oxidant
properties. These are important properties for future in vivo use.
Compound 1h displays similar protections to that of the two
reference molecules, NBA in the UV/H2O2 test and Trolox in
the Fenton test.
Concerning the three main series of pulvinic derivatives, the
two tests demonstrate that type II compounds 2 are the less
efficient protectors; good to excellent protections are obtained
with type I molecules 1 and similar or slightly higher protections
are observed with symmetrical derivatives 3. Inside the type II
series, almost no influence of the variation of the aromatic ring

Monoaromatic DeriVatiVes of PulVinic Acids
Table 1. Ring Opening Reaction of 14
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2457
Table 2. Tested Pulvinic Derivatives
substituent
compd
R1
R2
R3
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
2a
2b
2c
2d
3a
3b
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Ph
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2CH(CH3)2
CONH(CH2)3CH3
CON(CH2CH2)2O
CONH(CH2)3NH2
CON(CH3)OCH3
C(CH3)2OH
CO2Me
4-OMe Ph
4-Br Ph
4-F Ph
4-iPr Ph
styryl
4-OMe Ph
4-OMe Ph
4-OMe Ph
4-OMe Ph
4-OMe Ph
4-OMe Ph
Me
Me
Me
Me
Ph
4-OMe Ph
4-Br Ph
4-OMe Ph
Ph
CO2Me
CO2Me
CO2CH(CH3)2
CO2Me
CO2Me
4-OMe Ph
4-OMe Ph
protection activities in the same range, namely 30-48% in the
UV/H2O2 test and 37-98% in the Fenton test. However, in both
assays, the best protection results were obtained with the n-butyl
amide 1h. Finally, the tertiary alcohol 1l exhibited in both assays
a slightly lower potency than that of the best type I compounds
tested.
Scheme 3a
Symmetrical pulvinic derivatives 3a,b are very potent protec-
tors in both tests and equivalent to the best type I compound
1h.
In conclusion, results of UV/H2O2 and Fenton dThd protection
tests showed that the type I and symmetrical pulvinic derivative
tested are potent HO• scavengers as well as potent protectors
against an iron-based oxidative system.
a Reagents and conditions: MeLi (2 equiv), THF, -78 °C, 1 h.
Protection of Plasmid-DNA under Fenton Stress. To check
that the previous results were not limited to the simple thymidine
structural features, the protection of a supercoiled plasmid DNA
under Fenton-type oxidation and in the presence of selected
compounds was investigated. In the absence of protective agents
and under the optimized oxidative conditions, supercoiled DNA
(Figure 5, lane 1) is degraded, leading essentially to the
formation of linear DNA (Figure 5, lane 4, only small fractions
of supercoiled and circular DNA could be observed).
The protection efficiencies of the tested compounds were
characterized by the detection of significant remaining amounts
of supercoiled plasmid DNA after oxidation. As previously
reported, NBA displayed an opposite effect because it induced
a higher degree of plasmid degradation, which is an indication
of its pro-oxidant effect (Figure 5, lane 5-8).14 The symmetrical
derivative 3b and the type I product 1b efficiently protected
DNA from degradation (Figure 5, lane 9-12 and 13-16).
Moreover, a positive dose-response was observed, as the
proportion of supercoiled DNA increased with the concentration
of products tested. On the other hand, reaction performed in
the presence of type II product 2b did not show any significant
protection of DNA (Figure 7, compare lanes 4 and 17-20).
A larger set of compounds was studied for the DNA
protection under the same oxidative conditions (Figure 6).
Protectors were assayed at a single concentration (125 µM),
and results were expressed as the remaining % of supercoiled
DNA form (by light intensity integration of the three forms of
Figure 3. Structures of symmetrical pulvinic acids 3.
substitution or the ester nature is seen because compounds 2a-d
show similar results in each of the two dThd protection tests.
In the type I family, both dThd-based assays show similar
trends for the influence of the para aromatic substitution (1a-1f,
Figure 4). Indeed, by comparison with the nonsubstituted 1a
compound, only the para-methoxy substituent of 1b allows
conservation or slight improvement of the dThd protection. The
para aromatic substitution by a halogen (1c and 1d), an alkyl
(1e), or a vinyl chain (1f) decreases the protection effects on
both UV/H2O2 and Fenton tests.
Variations of the substitution of the exo-cyclic double bond
of type I compounds (1g-1l, Figure 4) lead also to important
variations in the protections, which are similarly observed in
both tests. Changing the methyl ester of 1b into an isopropyl
ester (1g) reduces to a large extent the protection efficiencies
(from 37% to 27% in the UV/H2O2 test and from 88% to 37%
in the Fenton test, Figure 4). Amides products 1h-1k displayed

2458 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Habrant et al.
Figure 4. (A) Protective effects of pulvinic derivatives under UV exposure: dThd protection assays. [dThd] ) 70 µM, [antioxidant] ) 100 µM,
H2O2 ) 5 mM in phosphate buffer 25 mM (pH 7.4), 254 nm, 1.75 J/cm2. Experiments were performed in duplicate. (B) Protective effects of
pulvinic derivatives under Fenton stress: dThd protection assays. [dThd] ) 70 µM, [antioxidant] ) 500 µM, Fe2+/EDTA/H2O2 (1:1:100) 700 mM
in phosphate buffer 25 mM (pH 7.4), 30 min. Experiments were performed in duplicate. Results are expressed in percent thymidine protection,
calculated by comparing results obtained in the presence and in the absence of protective agent.
Figure 5. DNA protection by pulvinic derivatives under Fenton stress. Oxidations of supercoiled plasmid pEGFPLuc (lane 1) were carried out in
the presence of 0.4 mM FeIISO4/EDTA/H2O2 (1:1:10) for 30 min. Control experiments were performed without iron (lane 2) or without H2O2 (lane
3). Oxidation of DNA in the absence (lane 4) or in the presence of four concentrations (500, 250, 125, 62.5 µM respectively) of antioxidants NBA
(lanes 5-8), 3b (lanes 9-12), 1b (lanes 13-16), and 2b (lanes 17-20). Linear, circular, and supercoiled DNA were separated by agarose gel
electrophoresis and stained with ethydium bromide.
potencies. For type I products, halogen-substituted derivatives
were less efficient than the nonsubstituted and the methoxy-
substituted products (Figure 6, compare products 1c and 1d to
1a and 1b) and the substitution of the exo-cyclic double bond
did not seem play a crucial role in this test, all products 1b, 1h,
1j, and 1l exhibiting around 40% of protection.
Briefly, protection of thymidine and of plasmid DNA showed
the same trend, indicating that type I products 1 were as efficient
as the symmetrical pulvinic acids 3. Type II products 2 protected
poorly these two biological targets. No pro-oxidant effect was
detected for types I and II and symmetrical compounds.
Scavenging of Superoxide Anion. The first product of
Figure 6. Protection of plasmidic-DNA by pulvinic derivatives (125
univalent reduction of oxygen is the superoxide anion O2•-. It
µM) under Fenton stress. The percentage of supercoiled plamid was
is generated in many biological processes,29,30 therefore com-
calculated by light intensity integration of the three forms of DNA in
•-
pounds able to scavenge O2 could be of great interest. To
assess the activity of our pulvinic derivatives for the scavenging
of O2•-, photoluminescence assays were performed using the
Photochem technique with standard kits ACL (Analytik Jena
electrophoresis gels: supercoiled, circular, and linear.
DNA in electrophoresis gels: supercoiled, circular, and linear).
Results are in very good agreement with the Fenton dThd
protection test because type II compounds 2a-2c were found
to be far less efficient protectors than type I 1a-1l and
symmetrical pulvinic derivatives 3a,b, which were of similar
AG).31,32 In these experiments, O2 is generated by optical
excitation (at 351 nm) of a methanolic solution of luminol used
as a photosensitizer; the same molecule is used as the radical
•-

Monoaromatic DeriVatiVes of PulVinic Acids
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2459
Figure 7. Suggested mechanism for the monoelectronic oxidation of pulvinic derivatives.
Table 3. Scavenging of Superoxide Anion
results obtained with the two thymidine protection assays and
the plasmid-DNA protection assay are very similar, demonstrat-
ing that the tested compounds retain their activity in different
oxidative stress conditions and toward different biological
targets. The three screening procedures clearly showed that type
I and symmetrical pulvinic derivatives display similar antioxi-
dant properties that are much higher than that of type II
molecules. This observation is supported by data in Figure 4
and Figure 5 for the two sets of molecules 1a-3a and 1b-3b.
We propose a simple model that allows rationalizing these
results (Figure 7). The first radical attack on phenolic antioxidant
is known to proceed via different mechanisms.35 The first main
type of mechanism involves a hydrogen atom transfer (HAT)
or a proton coupled electron transfer (PCET). The second main
type of oxidative mechanism involve a sequential proton loss
electron transfer (SPLET) and is known to take place under
specific reaction conditions when the phenolic antioxidant can
be deprotonated in the medium (Figure 7).
In the SPLET mechanism, the deprotonated phenolic function
usually reacts by electron transfer with the oxidative species,
faster than it does through a regular HAT mechanism. In the
experimental conditions used for the thymidine and DNA
protection assay (water buffered at physiological pH), and given
the reported pKa values for the enolic protons of pulvinic acids,36
the deprotonation of the enol is very favorable, and consequently
a SPLET mechanism should be preferred. Under these hypoth-
eses, a mechanistic model is suggested in Figure 7 (starting from
the deprotonated enols) in order to rationalize the results
obtained in the thymidine and DNA protection assays. The
prediction of simple mesomer forms give the indication that
the enol radicals generated on type I and symmetric derivatives
can be stabilized by delocalization on the ꢀ-cetoester fragment
of the lactone and on the south aromatic ring. In the case of
type II molecules, the delocalization is reduced because of the
absence of the south aromatic ring, which suggests that the
formation of this radical is less favorable.
compd
Trolox equiv (µg)
1b
1c
1g
1h
1i
141
113
105
86
79
81
87
77
54
1j
2c
2d
3b
detecting agent by luminol-enhanced chemiluminescence.33
Results were calculated as Trolox equivalent, which serves as
a reference for this test (Table 3). Expressed values correspond
to µg quantities of the tested compound needed to reach the
protection equivalent to 1 µg of Trolox.
Representative compounds of each type I, type II, and
symmetrical pulvinic acids were selected for this test. All tested
compounds are of similar efficiency but are less potent than
Trolox (in the range of 60-140 Trolox equivalents). Neverthe-
less, the scavenging of superoxide anion was detected in all
cases. Contrary to previous tests, no clear distinction is seen
here between the three series of type I, type II, and symmetrical
derivatives: 1b and 3b on one hand and 1g and 2d on the other
hand being of equivalent activity. The variation of the exo-cyclic
double bond substitution has also no influence on the measured
•-
O2 scavenging properties (1b and 1g-1j, Table 3).
Cytotoxicity. The in vitro toxicity of the newly synthesized
compounds was then measured on CHO cells using the MTT
colorimetric assay.34 Compounds were tested at 10, 30, and 100
µM, and none of the products, including type I products 1 (1a,
1c, 1i, 1h, 1l), type II 2 (2a, 2d), and symmetrical 3 (3b) showed
any noticeable toxicity with respect to the control culture.
Discussion
In this section, the various protection results will be compared
and a mechanistic hypothesis will be provided. Interestingly,

2460 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Habrant et al.
Thus the model also clarifies why the three type II compounds
2a-2c, differentiated by various para substituents on the north
aromatic ring, have similar and low activities in the two dThd
tests because this part is not expected to be involved in the first
radical reaction.
hydrogen bond and thus is more prone to be abstracted by the
oxidative species in the solution.
Complementary experiments are necessary in order to deter-
mine the precise antioxidant mechanisms involved in the
observed protection of thymidine, plasmid-DNA, and superoxide
anion by pulvinic derivatives. These studies will be performed
on selected compounds and will be published in due course.
Finally, the cytotoxicity results are very encouraging as a first
step before in-depth studies, as all tested compounds exhibited
the same pattern of harmlessness on CHO cells.
On the contrary, according to the model, the para substitution
of the south aromatic ring in type I compounds has an influence
on the protection activity. This is experimentally observed with
compounds 1a-1f on the dThd and plasmid DNA tests. Indeed,
we observed that a positive mesomer effect in the para position
(methoxy group, 1b) allows conservation or slight improvement
of the activity compared to the nonsubstituted compound 1a.
By comparison, a positive inductive effect (iso-propyl group,
1e), the presence of a halogen (1c and 1d), or the styryl
substitution (1f) is not favorable. To the best of our knowledge,
no study has described so far the influence of the substitutions
of the aromatic ring on the efficiency of the radical trapping
via a SPLET mechanism. However, our results are relatively
similar to studies on the HAT mechanism, where the influence
of the substitution of aromatic rings was related to the strength
of the BDE on the O-H bond of phenols. A para-methoxy is
known to stabilize the phenoxyl radical by conjugative electron
delocalization and to lower the BDE,19a while a para-halogen
strengthens it.37 Para-alkyl group are described to lower the
BDE but less effectively than para-methoxy.38 Contrarily, in
our case, compound 1e shows less satisfactory protection
potency than the nonsubstituted product 1a.
The influence of the substitution of the exo-cyclic double bond
on the antioxidant properties (1g-1l), which is clearly exhibited
in the dThd assays (Figure 4), is not explained by the proposed
model. These variations could modify the solubility properties
of the molecules or influence the intramolecular hydrogen bond
between the enolic proton and the carbonyl and thus the pKa
values. Nevertheless, the variations of activity highlight that this
is a key structural feature that needs to be optimized in order to
design a potent molecule. Despite a fine understanding, the
modification of the exo-cyclic double bond offered the most
active compound tested in the dThd and DNA protection assays,
namely the amide 1h.
Conclusion
From two previous accounts by our group, pulvinic acids
appeared to be very potent antioxidant agents, however very
little was known about the SAR that dictated this interesting
activity. To rapidly elucidate the main features of the SAR, our
strategy was to design simplified analogues of these natural
products. Following several synthetic strategies, 18 very diverse
pulvinic acids derivatives were successfully synthesized. These
molecules are divided into three structural categories: type I
and type II, which are monoaromatic derivatives, and the
symmetrical pulvinic derivatives.
Studies conducted on thymidine and plasmid DNA demon-
strated that type I and symmetrical derivatives are very potent
protectors of these biological targets against ROS (as efficient
as NBA) and a Fenton-type oxidative system. No pro-oxidant
effect was detected in the presence of iron, as it was observed
for the natural pigment, norbadione A. Because the type II
molecules were less potent, it can be concluded that the
antioxidant properties rely on the lactone part and the south
aromatic ring of pulvinic acids. SAR conclusions also highlight
the influence of the para substituent of the south aromatic ring
and of the substitution of the exo-cyclic double bond of pulvinic
acids. Among the newly synthesized molecules, compound 1h
was found to be the most active molecule.
Our results open the route to the synthesis of simplified
pulvinic acids such as type I molecules, with finely tuned
physicochemical properties (molecular weight, solubility) by
modulation of the phenyl and the exo-cyclic double bond
substitutions that will retain or even improve the antioxidant
properties of natural pulvinic acids. Moreover, the first dem-
onstration of the noncytotoxicity of type I compounds encourage
the study of biological applications of these molecules. Cur-
rently, amphiphilic pulvinic acid derivatives of type I are being
synthesized and studied as skin protectors via anti-inflammatory
effects or ROS scavenging. These dermocosmetic applications
will be reported in due course.
The complementary assay for the inhibition of O2•- was used
to determine whether the studied compounds are general free
radical scavengers or scavengers specific for the superoxide
anion.39 In several cases, a particular reactivity is associated to
superoxide anion compared to other ROS because of its lower
reactivity.40 Only moderate O2 scavenging activities were
•-
detected for our compounds in this assay, and the SAR main
features highlighted in the previous tests were not observed
(Table 3). Thus the radical trapping mechanisms on pulvinic
derivatives in this assay might differ from those happening in
the dThd and DNA protection assays. Indeed, we could expect
SPLET to electron rich superoxide anion to be less favorable
than to the electrophilic hydroxyl radicals. Under such hypoth-
esis, an HAT mechanism should be preferred but not expected
to be highly efficient for the pulvinic compounds because of
the presence of strong hydrogen bonding between the reactive
enolic hydrogen and the carbonyl function in pulvinic acids.
Such particular intramolecular hydrogen bonds are typical of
pulvinic derivatives and are present on each compound from
series 1, 2, and 3, which could explain the similar results
obtained for all pulvinic derivatives tested in the O2•- scavenging
test. Finally, the observed high potency of Trolox for the
Experimental Section
Chemistry. Flash column chromatographies were performed with
silica gel (40-63 µm). Nuclear magnetic resonance spectra (1H
and 13C) were recorded on a 200 or 300 MHz spectrometer equipped
with a DUAL probe. Melting points were uncorrected. For GC-
MS experiments, low-resolution mass spectra (chemical ionization)
and gas chromatography retention times were recorded using a
capillary column (25 mm × 0.22 mm) SGE BPX5 (5% phenyl
polysilphenylenesiloxane/95% methylpolysiloxane) with helium (29
mL/min; 113 kPa) and the following temperature conditions:
interface temperature 260 °C, detector temperature 302 °C, column
initial temperature 80 °C (2 min) increasing at the rate 25 °C/min.
Low and high resolution mass spectrometry (MS and HRMS,
respectively) data were obtained with an electrospray (ES) ion
source.
•-
scavenging of O2 could as well be due to a more efficient
Methyl 2-(2-Hydroxy-3-methoxy-5-oxo-2.5-dihydrofuran-2-
yl)propanoate (5). LDA was generated by addition of n-BuLi (1.5
M in hexanes, 10.2 mL, 15.30 mmol, 1.1 equiv) to a solution of
HAT phenomenon compared to pulvinic acids. Indeed the
phenolic proton in Trolox is not involved in an intramolecular

Monoaromatic DeriVatiVes of PulVinic Acids
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2461
1
iPr2NH (2.14 mL, 15.30 mmol, 1.1 equiv) in THF (60 mL) at -78
°C. The solution was warmed to -20 °C for 15 min and then cooled
back to -78 °C. A solution of methyl propionate (1.34 mL, 13.91
mmol, 1 equiv) in THF (10 mL) was added dropwise, and the
mixture was stirred at -78 °C for 45 min. Zinc chloride (1 M in
ether, 13.91 mL, 13.91 mmol, 1 equiv), THF (20 mL), and a
solution of methoxymaleic anhydride 420 (2.13 g, 16.69 mmol, 1.2
equiv) in THF (5 mL) were successively added. The mixture was
slowly allowed to warm to rt and stirred 2 h. Water was added,
and the aqueous phase was extracted with EtOAc. Aqueous phase
was then neutralized with saturated aqueous NH4Cl and extracted
twice with EtOAc. This extract was dried over anhydrous MgSO4
and concentrated to yield pure alcohol 5 (1.52 g, 51%) as a mixture
a diastereoisomers (70/30 by integration in 1H NMR). Major isomer:
1H NMR (200 MHz, CDCl3) δ 1.12 (d, J ) 7.1 Hz, 3H), 3.04 (q,
J ) 7.1 Hz, 1H), 3.79 (s, 3H), 3.91 (s, 3H), 5.11 (s, 1H). 13C NMR
(50 MHz, CDCl3) δ 11.2, 43.3, 53.2, 60.2, 90.7, 103.3, 170.0, 173.9,
white solid. H NMR (300 MHz, CDCl3) δ 2.14 (s, 3H), 3.71 (s,
3H), 3.81 (s, 3H), 3.82 (s, 3H), 6.93 (d, J ) 9.1 Hz, 2H), 7.41 (d,
J ) 9.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 15.0, 52.6, 55.4,
60.9, 108.8, 113.2, 114.0, 120.6, 131.2, 143.1, 160.1, 161.1, 168.0,
168.5. GC-MS: tr ) 11.0 min, MS 305 [M + H]+.
Methyl 2-(4-(4-Bromophenyl)-3-methoxy-5-oxofuran-2(5H)-
ylidene)propanoate (8c). The target compound was prepared from
7 and 4-bromophenylboronic acid using general procedure A. After
purification, product 8c was obtained (0.033 g, 63%) as a pale-
yellow solid. 1H NMR (200 MHz, CDCl3) δ 2.15 (s, 3H), 3.72 (s,
3H), 3.82 (s, 3H), 7.34 (d, J ) 8.5 Hz, 2H), 7.56 (d, J ) 8.5 Hz,
2H). 13C NMR (75 MHz, CDCl3) δ 15.2, 52.7, 61.3, 107.6, 114.3,
123.4, 127.6, 131.6, 131.7, 142.7, 162.0, 167.4, 168.3. GC-MS: tr
) 11.1 min, MS 353, 355 [M + H]+.
Methyl 2-(4-(4-Fluorophenyl)-3-methoxy-5-oxofuran-2(5H)-
ylidene)propanoate (8d). The target compound was prepared from
7 and 4-fluorophenylboronic acid using general procedure A. After
purification, product 8d was obtained (0.029 g, 67%) as a pale-
yellow solid. 1H NMR (300 MHz, CDCl3) δ 2.16 (s, 3H), 3.72 (s,
3H), 3.82 (s, 3H), 7.09-7.15 (m, 2H), 7.45-7.50 (m, 2H). 13C
NMR (75 MHz, CDCl3) δ 15.1, 52.6, 61.2, 108.1, 114.0, 115.6,
115.9, 124.6, 124.7, 131.9, 132.0, 142.9, 161.4, 161.8, 164.7, 167.6,
168.3. GC-MS: tr ) 9.8 min, MS 293 [M + H]+.
Methyl 2-(4-(4-Isopropylphenyl)-3-methoxy-5-oxofuran-2(5H)-
ylidene)propanoate (8e). The target compound was prepared from
7 and 4-iso-propylphenylboronic acid using general procedure A.
After purification, product 8e was obtained (0.027 g, 58%) as a
pale-yellow solid. 1H NMR (200 MHz, CDCl3) δ 1.25 (d, J ) 6.8
Hz, 6H), 2.15 (s, 3H), 2.91 (spt, J ) 6.8 Hz, 1H), 3.73 (s, 3H),
3.82 (s, 3H), 7.26 (d, J ) 8.3 Hz, 2H), 7.38 (d, J ) 8.3 Hz, 2H).
13C NMR (75 MHz, CDCl3) δ 15.1, 24.1, 34.1, 52.7, 61.1, 108.7,
113.3, 126.3, 127.5, 130.0, 143.0, 149.8, 163.8, 168.0, 168.5. GC-
MS: tr ) 11.0 min, MS 317 [M + H]+.
Methyl 2-(4-(E)-Styryl-3-methoxy-5-oxo-furan-2(5H)-ylidene)pro-
panoate (8f). The target compound was prepared from 7 and trans-
2-phenylvinylboronic acid using general procedure A. After
purification, product 8f was obtained (0.027 g, 61%) as a pale-
yellow solid. 1H NMR (300 MHz, CDCl3) δ 2.12 (s, 3H), 3.82 (s,
3H), 4.15 (s, 3H), 6.95 (d, J ) 16.1 Hz, 1H), 7.27-7.43 (m, 5H),
7.63 (d, J ) 16.1 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 15.4,
52.8, 61.7, 107.3, 113.7, 114.3, 127.0, 129.1, 135.5, 135.9, 137.1,
143.1, 160.1, 166.9, 167.5, 168.6. GC-MS: tr ) 11.3 min, MS 301
[M + H]+.
General Procedure B: Deprotection of the Enol Function. A
solution of compound 8 (1 equiv) was dissolved in DCM (15 mL/
mmol of 8) and cooled to 0 °C. A solution of BBr3 (1 M in DCM,
1 equiv) was slowly added. The solution was stirred 2 h at 0 °C
and then quenched by addition of 1 M HCl. After extraction with
EtOAc, the extracts were dried over anhydrous MgSO4 and
concentrated. The residue was purified by silica gel column
chromatography (DCM) to afford 1 as a yellow solid. Compounds
1a-f were prepared following general procedure B.
Methyl 2-(3-Hydroxy-5-oxo-4-phenylfuran-2(5H)-ylidene)pro-
panoate (1a). Yield: 85%. 1H NMR (300 MHz, CDCl3) δ 2.17 (s,
3H), 3.95 (s, 3H), 7.29-7.45 (m, 3H), 8.12 (d, J ) 6.0 Hz, 2H),
13.69 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 14.0, 54.3, 104.5,
111.9, 127.8, 128.2, 128.5, 129.3, 154.3, 160.2, 166.3, 172.1. HRMS
(ES): calcd for C14H13O5 261.0757 [M + H]+; found 261.0754.
Methyl 2-(3-Hydroxy-5-oxo-4-(4-methoxyphenyl)furan-2(5H)-
ylidene)propanoate (1b). Yield: 65%. 1H NMR (300 MHz, CDCl3)
δ 2.16 (s, 3H), 3.84 (s, 3H), 3.95 (s, 3H), 6.95 (d, J ) 8.8 Hz, 2H),
8.09 (d, J ) 8.8 Hz, 2H), 13.46 (s, 1H). 13C NMR (75 MHz, CDCl3)
δ 14.1, 54.4, 55.6, 104.6, 111.3, 114.2, 122.3, 129.4, 154.5, 159.3,
159.6, 166.9, 172.4. MS (ES): 289.2 [M - H]-. Elemental analysis
for C15H14O6: calcd, C, 62.07; H, 4.86; found C, 61.91; H, 4.89.
1
177.3. Minor isomer: H NMR (200 MHz, CDCl3) δ 1.36 (d, J )
7.1 Hz, 3H), 2.93 (q, J ) 7.1 Hz, 1H), 3.74 (s, 3H), 3.89 (s, 3H),
5.07 (s, 1H). 13C NMR (50 MHz, CDCl3) δ 12.3, 45.4, 52.9, 60.2,
90.3, 102.6, 169.5, 174.5, 178.8.
Methyl 2-(3-Methoxy-5-oxofuran-2(5H)-ylidene)propanoate
(6). A solution of alcohol 5 (0.643 g, 2.97 mmol, 1 equiv) in DCM
(30 mL) was cooled to 0 °C. Et3N (2.49 mL, 17.85 mmol, 6 equiv)
and DMAP (36 mg, 0.30 mmol, 0.1 equiv) were successively added.
A solution of TFAA (1.24 mL, 8.92 mmol, 3 equiv) in DCM (5
mL) was added dropwise, and the solution was stirred 2 h at 0 °C.
After addition of 1 M HCl, the mixture was extracted twice with
EtOAc. The extracts were dried over anhydrous MgSO4 and
concentrated. The residue was purified by silica gel column
chromatography (cyclohexane/EtOAc: 90/10 to 70/30) to afford 6
1
(0.525 g, 90%, E/Z: 90/10 by integration in H NMR) as a light-
1
yellow solid. E-isomer could be isolated. H NMR (300 MHz,
CDCl3) δ 2.08 (s, 3H), 3.77 (s, 3H), 3.88 (s, 3H), 5.28 (s, 1H). 13
C
NMR (75 MHz, CDCl3) δ 15.2, 52.7, 60.0, 91.2, 114.0, 143.9,
167.4, 168.0, 169.2. MS (ES): 199.1 [M + H]+.
Methyl 2-(4-Iodo-3-methoxy-5-oxofuran-2(5H)-ylidene)pro-
panoate (7). To a solution of 6 (0.116 g, 0.59 mmol, 1 equiv) in
MeCN (12 mL) were successively added iodine (0.45 g, 1.76 mmol,
3 equiv) and CAN (0.97 g, 1.76 mmol, 3 equiv) and the mixture
was heated at 40 °C for 3 h. After addition of saturated aqueous
Na2S2O3, the mixture was extracted three times with EtOAc. The
extracts were washed with saturated aqueous Na2S2O3 and brine,
dried over anhydrous MgSO4, and concentrated. The residue was
purified by silica gel column chromatography (cyclohexane/EtOAc:
1
90/10 to 80/20) to afford 7 (0.197 g, 94%) as a white solid. H
NMR (300 MHz, CDCl3) δ 2.05 (s, 3H), 3.77 (s, 3H), 4.34 (s,
3H). 13C NMR (75 MHz, CDCl3) δ 15.1, 52.2, 52.9, 60.6, 114.2,
144.0, 165.9, 167.1, 168.0. GC-MS: tr ) 8.8 min, MS 325 [M +
H]+.
General Procedure A: Suzuki-Miyaura cross-coupling with
7; Methyl 2-(3-methoxy-5-oxo-4-phenyl-2(5H)-ylidene)pro-
panoate (8a). Iodinated compound 7 (0.042 g, 0.15 mmol, 1 equiv),
phenylboronic acid (0.18 mmol, 1.2 equiv), supported catalyst23
(0.007 g, 0.7 mol%), and iPr2NH (0.050 mL, 0.37 mmol, 2.5 equiv)
were dissolved in MeCN (3 mL) and H2O (1 mL). The solution
was degassed under an Ar flow for 1 h and then heated at 85 °C
for 6 h 30 min. The mixture was then filtered on celite. After
addition of H2O, the mixture was extracted with EtOAc. The
extracts were washed with H2O, dried over anhydrous MgSO4, and
concentrated. The residue was purified by silica gel column
chromatography (cyclohexane/EtOAc: 80/20) to afford 8a (0.028
1
g, 78%) as a white solid. H NMR (300 MHz, CDCl3) δ 2.16 (s,
3H), 3.72 (s, 3H), 3.82 (s, 3H), 7.36-7.46 (m, 5H). 13C NMR (75
MHz, CDCl3) δ 14.9, 52.4, 61.1, 108.3, 113.5, 128.3, 128.5, 128.8,
130.0, 142.8, 161.5, 167.6, 168.2. GC-MS: tr ) 9.5 min, MS 276
[M + H]+.
Methyl 2-(3-Hydroxy-5-oxo-4-(4-bromophenyl)furan-2(5H)-
ylidene)propanoate (1c). Yield: 58%. 1H NMR (300 MHz, CDCl3)
δ 2.18 (s, 3H), 3.98 (s, 3H), 7.54 (d, J ) 8.8 Hz, 2H), 8.03 (d, J
) 8.8 Hz, 2H), 13.81 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 14.0,
54.4, 103.6, 112.4, 122.3, 128.9, 129.3, 131.7, 154.2, 160.5, 166.0,
Methyl 2-(4-(4-Methoxyphenyl)-3-methoxy-5-oxofuran-2(5H)-
ylidene)propanoate (8b). The target compound was prepared from
7 and 4-methoxyphenylboronic acid using general procedure A.
After purification, product 8b was obtained (0.030 g, 65%) as a

2462 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Habrant et al.
172.2. HRMS (ES): calcd for C14H12BrO5 338.9854, 340.9844 [M
+ H]+; found 338.9863, 340.9842.
afforded product 13a (0.055 g, 18%), E-isomer, as a pale-yellow
1
solid. H NMR (300 MHz, CDCl3) δ 2.14 (s, 3H), 3.29 (s, 3H),
5.40 (s, 2H), 7.32-7.60 (m, 10H). 13C NMR (75 MHz, CDCl3) δ
9.3, 52.3, 74.4, 101.8, 115.4, 128.5, 128.8, 129.0, 129.2, 129.3,
131.3, 134.6, 141.4, 161.3, 166.8, 169.9. MS (ES): 351.1 [M +
H]+.
Methyl 2-(3-Hydroxy-5-oxo-4-(4-fluorophenyl)furan-2(5H)-
ylidene)propanoate (1d). Yield: 71%. 1H NMR (300 MHz, CDCl3)
δ 2.18 (s, 3H), 3.98 (s, 3H), 7.08-7.14 (m, 2H), 8.11-8.16 (m,
2H), 13.71 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 14.0, 54.4, 103.8,
112.1, 115.4, 115.7, 125.4, 129.8, 154.3, 159.6, 160.7, 164.0, 166.3,
172.2. HRMS (ES): calcd for C14H12FO5 279.0663 [M + H]+; found
279.0657.
Methyl 2-(3-Hydroxy-5-oxo-4-(4-iso-propylphenyl)furan-
2(5H)-ylidene)propanoate (1e). Yield: 69%. 1H NMR (300 MHz,
CDCl3) δ 1.26 (d, J ) 7.0 Hz, 6H), 2.18 (s, 3H), 2.93 (spt, J ) 7.0
Hz, 1H), 3.96 (s, 3H), 7.29 (d, J ) 8.5 Hz, 2H), 8.03 (d, J ) 8.5
Hz, 2H), 13.57 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 13.9, 24.0,
34.2, 54.3, 104.9, 111.6, 126.6, 126.7, 127.9, 149.2, 154.5, 159.6,
166.4, 172.2. HRMS (ES): calcd for C17H19O5 303.1227 [M + H]+;
found 303.1220.
Methyl 2-(3-(Benzyloxy)-4-methyl-5-oxofuran-2(5H)-ylidene)-
2-(4-bromo)phenylacetate (13c). Following the procedure de-
scribed for compound 6, dehydratation of alcohol 12c (0.089 g,
0.20 mmol) afforded product 13c (0.038 g, 44%) as a white solid,
1
mixture of E and Z forms. E-isomer could be isolated. H NMR
(200 MHz, CDCl3) δ 2.14 (s, 3H), 3.27 (s, 3H), 5.40 (s, 2H),
7.31-7.38 (m, 2H), 7.42-7.52 (m, 8H). 13C NMR (50 MHz,
CDCl3) δ 9.3, 52.4, 74.5, 102.2, 114.3, 123.6, 127.3, 128.7, 129.0,
129.2, 130.3, 131.1, 132.0, 141.9, 161.2, 166.5, 169.6. MS (ES,
pos): 429.2, 431.2 [M + H]+.
Methyl 2-(3-Hydroxy-4-methyl-5-oxofuran-2(5H)-ylidene)-2-
phenylacetate (2a). To a solution of compound 12a (0.121 g, 0.35
mmol) in DCM (10 mL) was added Pd/C (10%, 0.17 g, 0.1 equiv).
H2 was then introduced and the mixture was stirred overnight at rt.
After filtration on celite and concentration, the residue was purified
by silica gel column chromatography (DCM) to afford product 2a
Methyl 2-(3-Hydroxy-5-oxo-4-(E)-styrylfuran-2-(5H)-ylidene)pro-
1
panoate (1f). Yield: 61%. H NMR (200 MHz, CDCl3) δ 2.15 (s,
3H), 3.96 (s, 3H), 6.88 (d, J ) 16.5 Hz, 1H), 7.26-7.35 (m, 3H),
7.50-7.53 (m, 3H), 7.58 (d, J ) 16.1 Hz, 1H), 13.34 (s, 1H). 13
C
NMR (50 MHz, CDCl3) δ 13.9, 54.2, 105.3, 111.7, 114.7, 126.8,
128.3, 128.8, 133.7, 137.4, 154.2, 159.0, 166.2, 172.0. HRMS (ES):
calcd for C16H15O5 287.0914 [M + H]+; found 287.0907.
4-Benzyloxy-3-methylfuran-2(5H)-one (10). Tetronic derivative
1
(0.046 g, 48%) as a white solid. H NMR (200 MHz, CDCl3) δ
1.90 (s, 3H), 3.85 (s, 3H), 7.21-7.27 (m, 2H), 7.38-7.43 (m, 3H),
12.86 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 6.6, 54.3, 104.4, 114.8,
128.1, 128.5, 130.0, 132.0, 155.2, 161.5, 168.0, 171.4. HRMS (ES):
calcd for C14H13O5 261.0757 [M + H]+; found 261.0753.
9
25 (0.932 g, 8.17 mmol, 1 equiv) was suspended in THF (40 mL).
Benzyl alcohol (1.01 mL, 9.80 mmol, 1.2 equiv) and PPh3 (2.57 g,
9.80 mmol, 1.2 equiv) were successively added. The solution was
cooled to 0 °C and DIAD (1.94 mL, 9.80 mmol, 1.2 equiv) was
slowly added and the mixture was stirred overnight at rt. After
concentration, the residue was purified by silica gel column
chromatography (DCM) to yield compound 10 (1.23 g, 74%), as a
pale-yellow solid. 1H NMR (200 MHz, CDCl3) δ 1.86 (s, 3H), 4.63
(s, 2H), 5.22 (s, 2H), 7.33-7.46 (m, 5H). 13C NMR (75 MHz,
CDCl3) δ 7.2, 65.9, 72.1, 99.3, 127.3, 128.8, 135.0, 171.5, 175.2.
MS (ES): 205.1 [M + H]+.
Methyl 2-(4-Bromophenyl)-2-(3-hydroxy-4-methyl-5-oxofu-
ran-2(5H)-ylidene)acetate (2c). Following the procedure described
for compound 2a, catalytic hydrogenation of 12c (0.035 g, 0.08
mmol) afforded compound 2c (0.018 g, 66%) as a white solid. 1H
NMR (200 MHz, CDCl3) δ 1.89 (s, 3H), 3.85 (s, 3H), 7.09 (d, J )
8.6 Hz, 2H), 7.52 (d, J ) 8.6 Hz, 2H), 12.79 (s, 1H). MS (ES):
339.0, 341.0 [M + H]+. HRMS (ES): calcd for C14H11BrO5
338.9863, 340.9844 [M + H]+; found 338.9872, 340.9858.
Methyl 2-(3-Hydroxy-4-methyl-5-oxofuran-2(5H)-ylidene)2-
(4-methoxyphenyl)acetate (2b). A solution of dilactone 1426 (35.4
mg, 0.14 mmol) in MeOH (5 mL) was heated under reflux
overnight. After concentration, the residue was purified by silica
gel column chromatography (cyclohexane/AcOEt: 80/20 to 70/30)
to afford product 1c (21.9 mg, 54%) as a yellow solid and product
2c (17.9 mg, 44%) as a white solid. 2b: 1H NMR (300 MHz, CDCl3)
δ 1.88 (s, 3H), 3.83 (s, 3H), 3.85 (s, 3H), 6.91 (d, J ) 8.7 Hz, 2H),
7.16 (d, J ) 8.8 Hz, 2H), 12.84 (s, 1H). 13C NMR (75 MHz, CDCl3)
δ 6.6, 54.3, 55.4, 104.4, 113.7, 114.7, 124.2, 131.5, 155.0, 159.8,
161.6, 168.2, 171.7. HRMS (ES): calcd for C15H15O6 291.0863 [M
+ H]+; found 291.0865.
Iso-propyl 2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-
2(5H)-ylidene)propanoate (1g) and Iso-propyl 2-(3-Hydroxy-4-
methyl-5-oxofuran-2(5H)-ylidene)2-(4-methoxy phenyl)acetate
(2d). A solution of dilactone 1426 (18.8 mg, 0.07 mmol) in i-PrOH
(3 mL) was heated under reflux overnight. After concentration, the
residue was purified by silica gel column chromatography (cyclo-
hexane/AcOEt: 80/20 to 70/30) to afford product 1g (13.4 mg, 58%)
as a yellow solid and product 2d (8.9 mg, 40%) as a white solid.
1g: 1H NMR (300 MHz, CDCl3) δ 1.38 (d, J ) 6,4 Hz, 6H), 2.14
(s, 3H), 3.83 (s, 3H), 5.21 (spt, J ) 6.4 Hz, 1H), 6.95 (d, J ) 9.1
Hz, 2H), 8.10 (d, J ) 9.1 Hz, 2H), 13.74 (s, 1H); 13C NMR (75
MHz, CDCl3) δ 13.9, 21.7, 55.3, 72.0, 104.3, 112.1, 113.9, 122.1,
129.2, 154.2, 158.9, 159.3, 166.7, 171.2; HRMS (ES): calcd for
C17H19O6 [M + H]+ 319.1149; found 319.1167. 2d: 1H NMR (300
MHz, CDCl3) δ 1.24 (d, J ) 6.4 Hz, 6H), 1.87 (s, 3H), 3.83 (s,
3H), 5.18 (spt, J ) 6.4 Hz, 1H), 6.89 (d, J ) 8.7 Hz, 2H), 8.10 (d,
J ) 8.7 Hz, 2H), 13.03 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 6.6,
21.5, 55.4, 72.1, 103.9, 113.4, 115.6, 124.4, 131.6, 154.4, 159.6,
161.8, 168.4, 170.7; MS (ES): 319.1 [M + H]+; HRMS (ES): calcd
for C17H19O6 319.1176 [M + H]+; found 319.1188.
Methyl 2-(3-(Benzyloxy)-4-methyl-5-oxo-2.5-dihydrofuran-2-yl)-
2-hydroxy-2-phenyl Acetate (12a). LDA was generated by addition
of n-BuLi (1.5 M in hexanes, 3.74 mL, 5.61 mmol, 1.5 equiv) to
a solution of iPr2NH 0.79 mL, 5.61 mmol, 1.5 equiv) in THF (20
mL) at -78 °C. The solution was warmed to -20 °C for 15 min
and then cooled back to -78 °C. A solution of 9 (0.764 g, 3.74
mmol, 1 equiv) in THF (10 mL) was slowly added. After 30 min,
ketoester 11a (1.6 mL, 11.23 mmol, 3 equiv) was added. The
mixture was then allowed to warm to rt. After addition of saturated
aqueous NH4Cl, the mixture was extracted with EtOAc. The extract
was dried over anhydrous MgSO4 and concentrated. The residue
was purified by silica gel column chromatography (cyclohexane/
AcOEt: 90/10 to 100% AcOEt) to afford alcohol 12a (0.950 g,
69%) as a yellow oil, mixture of diastereoisomeric forms (65/35
1
1
by integration in H NMR). Major isomer could be isolated. H
NMR (200 MHz, DMSO-d6): δ: 1.94 (s, 3H), 3.29 (s, 3H), 5.47
(dd, J ) 11.0 Hz, J ) 4.2 Hz, 2H), 5.75 (s, 1H), 6.36 (s, 1H),
7.33-7.44 (m, 8H), 7.62 (d, J ) 7.8 Hz, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 8.5, 52.1, 72.9, 77.3, 80.1, 98.4, 126.1, 127.8, 128.0,
128.1, 128.3, 128.4, 135.7, 139.3, 169.5, 171.3, 174.1. MS (ES):
391.0 [M + Na]+.
Methyl 2-(3-(benzyloxy)-4-methyl-5-oxo-2.5-dihydrofuran-2-
yl)-2-(4-bromophenyl)-2-hydroxyacetate (12c). The target com-
pound was prepared from 9 (0.226 g, 1.11 mmol, 1 equiv) and
ketoester 11c (0.807 mL, 11.23 mmol, 3 equiv), following the
procedure described for compound 12a. Purification of the residue
(cyclohexane/AcOEt: 90/10 to 50/50) afforded alcohol 12c (0.329
g, 69%) as a yellow oil, equimolar mixture of diastereoisomeric
forms (1H NMR). 1H NMR (200 MHz, CDCl3, mixture) δ 2.02 (s,
3H), 2.06 (s, 3H), 3.37 (s, 3H), 3.86 (s, 3H), 5.12 (s, 2H), 5.34 (s,
2H), 5.41 (s, 1H), 6.70 (s, 1H), 7.27-7.55 (m, 18H). MS (ES):
447.1, 449.0 [M + H]+.
N-Butyl-2-(3-hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-
ylidene)propanamide (1h). To a solution of dilactone 1426 (23.6
mg, 0.09 mmol, 1equiv) in 1.5 mL of THF was slowly added a
solution of TBAF (1 M solution in THF, 0,18 mL, 0,18 mmol, 2
Methyl 2-(3-(Benzyloxy)-4-methyl-5-oxofuran-2(5H)-ylidene)-
2-phenylacetate (13a). Following the procedure described for
compound 6, dehydratation of alcohol 12a (0.356 g, 0.89 mmol)

Monoaromatic DeriVatiVes of PulVinic Acids
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2463
equiv) at -35 °C. The mixture was stirred for 15 min and then
cooled to -78 °C. A solution of butylamine (23 µL, 0.22 mmol,
2.5 equiv) in 1 mL of THF was added. After 15 min of stirring at
-78 °C, the mixture was allowed to warm to room temperature.
After concentration in vacuo, the crude residue was purified by
chromatography on silica gel (DCM) to furnish amide 1h (27.4
mg, 83%) as an orange solid. 1H NMR (200 MHz, CDCl3) δ 0.97
(t, J ) 7.4 Hz, 3H), 1.65-1.25 (m, 4H), 2.18 (s, 3H), 3.50-3.40
(m, 2H), 3.83 (s, 3H), 6.32 (brs, 1H), 6.95 (d, J ) 9.0 Hz, 2H),
8.11 (d, J ) 9.0 Hz, 2H), 15.82 (s, 1H). 13C NMR (50 MHz, CDCl3)
δ 13.7, 13.8, 20.2, 31.2, 40.8, 55.4, 102.8, 111.3, 113.9, 122.6,
128.9, 152.9, 159.1, 160.8, 167.5, 169.0. HRMS (ES) calcd for
C18H22NO5 [M + H]+ 332.1466; found 332.1480.
140.9, 160.0, 162.0, 170.8. MS (ES): 291.1 [M + H]+. HRMS (ES):
calcd for C16H19O5 291.1227 [M + H]+; found 291.1224.
Biology. Thymidine Protection Assay under UV Exposure
and Fenton Stress. These procedures have been previously
described.14
Scavenging of Superoxide Anion. Antioxidant capacity was
mesured by Photochem apparatus from Analytik Jena. ACL kits
of reagents (ref 400-803 from Analytik Jena) were used as well
as PCL soft to enable results analysis: kinetic and nanomol
equivalent of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid) calculated by integration of the curves on a typical
interval of 180 s. Results are expressed in µg of compound
equivalent to the activity of 1 µg of standard (Trolox).
(E)-4-Hydroxy-3-(4-methoxyphenyl)-5-(1-morpholino-1-oxo-
propan-2-ylidene)furan-2(5H)-one (1i). To a solution of dilactone
1426 (25.5 mg, 0.10 mmol, 1equiv) in 1.5 mL of THF was slowly
added a solution of TBAF (1 M solution in THF, 0,20 mL, 0,20
mmol, 2 equiv) at -35 °C. The mixture was stirred 15 min and
then cooled to -78 °C. A solution of morpholine (22 µL, 0.25
mmol, 2.5 equiv) in 1 mL of THF was added. After 15 min of
stirring at -78 °C, the mixture was allowed to warm to room
temperature. After concentration in vacuo, the crude residue was
purified by chromatography on silica gel (DCM) to furnish amide
1i (32.9 mg, 96%) as a yellow solid. 1H NMR (300 MHz, CDCl3)
δ 2.15 (s, 3H), 3.63-3.70 (m, 4H), 3.70-3.75 (m, 4H), 3.83 (s,
3H), 6.94 (d, J ) 9.1 Hz, 2H), 8.04 (d, J ) 9.1 Hz, 2H). 13C NMR
(75 MHz, CDCl3) δ 16.3, 46.2, 55.4, 66.9, 104.6, 112.6, 114.0,
121.9, 129.2, 150.3, 158.8, 159.3, 166.9, 171.2. HRMS (ES) calcd
for C18H20NO6 [M + H]+ 346.1285; found 346.1291.
Acknowledgment. This work was supported by the Centre
National de la Recherche Scientifique, Laboratoires Pierre Fabre
Dermo-Cosme´tique and De´le´gation Ge´ne´rale pour l’Armement.
We are grateful to Dr. Guy Zuber for providing plasmid
pEGFPLuc and for helpful discussions. We are grateful to Herve´
Volland, Marie-Claire Nevers, and Christophe Cre´minon from
CEA DSV/iBiTec-S/SPI for providing the anti-dThd monoclonal
antibody and for experimental support.
Supporting Information Available: Detailed descriptions of
HPLC methods and purity values of final compounds; EC50
measurements for selected compounds in the protection of thymi-
dine assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
N-(3-Aminopropyl)-2-(3-hydroxy-4-(4-methoxyphenyl)-5-oxo-
furan-2(5H)ylidene) Propanamide (1j). To a solution of dilactone
1426 (89.1 mg, 0.34 mmol, 1 equiv) in DCM (3 mL) was added
1.3-diaminopropane (0,14 mL, 0.17 mmol, 0.5 equiv) and the
solution was stirred overnight at rt. After concentration, the residue
was purified by silica gel column chromatography (DCM/MeOH:
100/0 to 80/20) to afford product 21 (36 mg, 64%), as a yellow
solid. 1H NMR (200 MHz, MeOD) δ 1.87-1.97 (m, 2H), 2.07 (s,
3H), 3.03 (t, J ) 7.3 Hz, 2H), 3.42 (t, J ) 6.1 Hz, 2H), 3.78 (s,
3H), 6.86 (d, J ) 9.0 Hz, 2H), 7.95 (d, J ) 9.0 Hz, 2H). MS (ES):
333.0 [M + H]+. HRMS (ES): calcd for C17H21N2O5 333.1445 [M
+ H]+; found 333.1449.
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JM801500H